Anoectochilus roxburghii Extract Extends the Lifespan of Caenorhabditis elegans through Activating the daf-16/FoxO Pathway

As a significant global issue, aging is prompting people’s interest in the potential anti-aging properties of Anoectochilus roxburghii (A. roxburghii), a plant traditionally utilized in various Asian countries for its purported benefits in treating diabetes and combating aging. However, the specific anti-aging components and mechanisms of A. roxburghii remain unclear. This study aims to investigate the anti-aging effects and mechanisms of A. roxburghii extract E (ARE). Caenorhabditis elegans (C. elegans) were exposed to media containing different concentrations of ARE whose superior in vitro radical scavenging capacity was thus identified. Lifespan assays, stress resistance tests, and RT-qPCR analyses were conducted to evaluate anti-aging efficacy, reactive oxygen species (ROS) levels, antioxidant enzyme activity, and daf-16, sod-3, and gst-4 levels. Additionally, transcriptomic and metabolomic analyses were performed to elucidate the potential anti-aging mechanisms of ARE. Fluorescence protein assays and gene knockout experiments were employed to validate the impacts of ARE on anti-aging mechanisms. Our results revealed that ARE not only prolonged the lifespan of C. elegans but also mitigated ROS and lipofuscin accumulation, and boosted resistance to UV and heat stress. Furthermore, ARE modulated the expression of pivotal anti-aging genes including daf-16, sod-3, and gst-4, facilitating the nuclear translocation of DAF-16. Significantly, ARE failed to extend the lifespan of daf-16-deficient C. elegans (CF1038), indicating its dependency on the daf-16/FoxO signaling pathway. These results underscored the effectiveness of ARE as a natural agent for enhancing longevity and stress resilience to C. elegans, potentially to human.


Introduction
Aging is a complex biological process characterized by the gradual decline in the physiological functions of tissues and organs, as well as a loss of repair capabilities, which increases the risk to various diseases [1][2][3].To delay aging, several strategies have been proposed, including calorie restriction, regular exercise, and dietary supplementation [4,5].Recently, there has been a marked increase in interest in identifying natural compounds that can delay aging and extend lifespan.Among the range of natural products, medicinal E. coli OP50 and C. elegans (N2, TJ356, CF1553, CL2166, CF1038) were acquired from the Caenorhabditis Genetics Center (CGC, Minneapolis, MN, USA). A. roxburghii was provided from the Jinhua Academy of Agricultural Sciences (Jinhua, Zhejiang, China).Plant specimens were preserved at the Southern Agriculture and Forestry Resources Laboratory of Xingzhi College, Zhejiang Normal University.The following reagents were utilized in the experiments: agar, KH 2 PO 4 , Na 2 HPO 4 , NaH 2 PO 4 , NaCl, NaClO, CaCl 2 , NaOH, MgSO 4 , cholesterol, yeast extract, tryptone, and peptone were purchased from Sangon Biotech (Shanghai, China).TB Green ® Premix Ex Taq™ and PrimeScript™ RT reagent Kit with gDNA Eraser were supplied by Takara (Beijing, China).Commercial kits for measuring SOD, MDA, CAT, and GSH-px were provided by the Nanjing Institute of Biological Engineering (Nanjing, China).Reactive Oxygen Species Assay Kit and Detergent Compatible Bradford Protein Assay Kit were obtained from Beyotime (Shanghai, China).A BCA assay kit was purshased from Nanjing Institute of Biological Engineering (Nanjing, China).

Preparation and Identification of AREs
The A. roxburghii were divided into five categories based on different polarities: A, B, C, D, and E. The detailed methods for extraction and screening are provided in Appendix A. The A. roxburghii fraction E, named ARE, had exhibited superior antioxidant properties compared to other fractions during in vitro antioxidant experiments.Subsequently, the LC-MS was used to identify the components of ARE and the condition as follows.
After freeze-drying, 8 mg ARE were weighed into a 1.5 mL EP tube, two small steel balls were added, and 400 µL methanol-water (v:v = 4:1, including mixed internal standard, 4 µg/mL) was added.After pre-cooling for 2 min in the −40 • C refrigerator, it was put it into the grinder for grinding (60 Hz, 2 min).It then underwent ultrasonic extraction in an ice water bath for 10 min and was left overnight at −40 • C. Then the sample was centrifuged for 10 min (12,000 rpm, 4 • C), extracted 150 µL supernatant with a syringe, filtered with 0.22 µm organic phase pinhole filter, transfered to LC sample vial, and stored at −80 • C until LC-MS analysis.
The mass spectra were acquired ranging from 70 to 1050 m/z with a primary resolution of 70,000 and a secondary resolution of 17,500.The sheath gas flow rate was 35, and the auxiliary gas flow rate was 8.The spray voltages were set at 3.8 kV (positive ion) and 3 kV (negative ion).The capillary temperature was maintained at 320 • C, and the auxiliary gas heater temperature was at 350 • C. Progenesis QI v3.0 software (Nonlinear Dynamics, Newcastle, UK) was used for baseline filtering, peak recognition, integration, retention time correction, peak alignment, and normalization of the original data.

Culture and Treatment of C. elegans
The C. elegans were cultured at 20 • C in nematode growth media (NGM) with E. coli OP50 serving as the food source and treated with sodium hypochlorite to synchronize the populations.The protocols for cultivation and maintenance followed those outlined in the WormBook [25].The dosed plates were prepared by adding 10 µg/mL and 100 µg/mL ARE into the NGM and then coated with OP50 on the surface and incubated at 37 • C for 12 h.Subsequently, synchronized C. elegans (L1 stage) were cultured at 20 • C for 3 days in NGM with OP50, water, 10 µg/mL, and 100 µg/mL ARE to evaluate the toxicity of ARE.The body length, locomotion, and pumping rates of the C. elegans were measured using an Mshot ML31 biomicroscope with an MShot Image Analysis System (Guangzhou Micro-shot Technology Co., Guangzhou, China).Statistical analysis was performed using a Student's t-test.The data are presented as mean ± SD.
Furthermore, to evaluate the preference of C. elegans for OP50 mixed with ARE, 50 µL of OP50 alone and 50 µL of OP50 mixed with 100 µg/mL ARE were seeded on opposite sides of an NGM plate.Then, 200 worms were placed in the center of the plate.After 1 h, the number of worms on each bacterial lawn was counted.This experiment was repeated three times.

Lifespan Analysis
Synchronized L4 stage C. elegans were picked and placed on standard NGM plates containing different concentrations of ARE, with ample OP50 provided as food.The NGM plates also contained 100 µM 5-fluoro-2-deoxyuridine (FUDR).The C. elegans were observed every 24 h until all had died.
After culturing synchronized L4 stage C. elegans for 2 days, they were subjected to UV irradiation and heat treatment at 37 • C to induce stress responses, which differ slightly from conventional lifespan assays.The UV treatment group was monitored every 24 h, whereas the heat treatment group was observed every hour until all had died.The data were statistically analyzed using the log-rank test.

Lipofuscin Level Assay
L4 stage larvae were incubated with 0 µg/mL, 10 µg/mL, or 100 µg/mL ARE.In 7-day-old worms, the autofluorescence of intestinal lipofuscin was measured through the use of a fluorescence microscopy (Guangzhou Micro-shot Technology Co., Guangzhou, China).The fluorescence intensity of 20 worms was quantified using the Image J software (version 15.4f) to determine lipofuscin levels.

Stress Tolerance Tests
The L4 stage C. elegans were treated with water, 10 µg/mL or 100 µg/mL ARE for 48 h, and then randomly divided into the control and treated groups with 200 worms per group.Afterward, the C. elegans were exposed to 100 mj of energy under a UV for 5 min to evaluate the UV protection by ARE.
In addition, the C. elegans were incubated at 37 • C, and their death number was recorded hourly to evaluate the resistance to heat stress of ARE.
In each experiment, death was determined by a lack of response to touch with a platinum wire.Unexpected deaths, such as those from loss or wall climbing, were excluded.Statistical analysis of the data was performed using the log-rank test.

Determination of ROS and Antioxidant Enzymes
Synchronized L4 stage C. elegans were treated in the NGM containing water, 10 µg/mL and 100 µg/mL ARE for 48 h, respectively.Subsequently, M9 buffer was used to wash C. elegans 3 times.Then, the active oxygen probe (DCFH-DA) was added at a ratio of 1:1000 and incubated at 20 • C for 25 min.After incubation, C. elegans were washed three times with M9 buffer and then placed on 2% agarose pads for photographing.ROS levels were measured using a microplate reader (Spark03030923, Tecan, Mannedorf, Switzerland) at an excitation wavelength of 488 nm and an emission wavelength of 525 nm.
Synchronized L4 stage C. elegans were transferred onto NGM containing 0 µg/mL, 10 µg/mL, and 100 µg/mL ARE, respectively.A total of 500 C. elegans were selected and washed with M9 buffer to remove residual OP50.The supernatant was then collected by grinding, crushing, and centrifugation (3000 rpm, 10 min) in an ice bath.The levels of SOD, CAT, and GSH-px were measured following the instructions of the commercial kits.Subsequently, protein concentrations were determined using a BCA assay kit.Statistical analysis was performed using the Student's t-test.The data are presented as mean ± SD.

RNA-Seq
The L4 stage worms were cultured on NGM plates containing 100 µg/mL ARE at 20 • C for 48 h.Subsequently, they were washed with M9 buffer to remove OP50, then flash-frozen in liquid nitrogen and stored at −80 • C. Afterwards, they were sent to OE Biotech (Shanghai, China) for subsequent library construction analysis.Alignment to the C. elegans reference genome was performed using HISAT2 software (version 2.1.0)to calculate gene expression levels (FPKM).DESeq2 software (version 1.22.2) was used to conduct differential expression gene (DEG) analysis, with DEGs defined as genes having a p-value <0.05 and a fold change >2 or <0.5.

Metabonomics
The L4 stage worms were cultured on NGM plates containing 100 µg/mL ARE at 20 • C for 48 h.Subsequently, they were washed off with M9 buffer to remove OP50, then flash-frozen in liquid nitrogen and stored at −80 • C. Afterwards, they were sent to OE Biotech (Shanghai, China) for subsequent differential metabolite analysis.Metabolites were identified based on multiple dimensions including retention time (RT), accurate mass, secondary fragmentation, and isotope distribution using The Human Metabolome Database (HMDB), Lipidmaps (v2.3),METLIN database, and the LuMet-Animal3.0 local database.Statistical significance (p-value) was computed using a single-factor analysis (t-test).Metabolites with VIP > 1 and a p-value < 0.05 were considered differentially expressed metabolites (DEMs).

Real-Time Quantitative PCR
The worms were treated with 100 µg/mL ARE for 2 days, and total RNA was extracted from the worms using the RNAiso Plus kit (Takara, Dalian, China) following the manufacturer's instructions.Possible genomic DNA contamination in the extracted RNA was removed using the PrimeScript™ RT reagent kit with gDNA Eraser kit (Takara, Dalian, China), and then the RNA was reverse transcribed into cDNA using the same PrimeScript™ RT reagent kit with gDNA Eraser.Subsequently, real-time quantitative PCR was performed on the cDNA obtained from C. elegans treated with 100 µg/mL ARE, using the TB Green ® Premix Ex Taq™ kit (Takara, Dalian, China).The relative gene expression was calculated using the 2 −∆∆Ct method, with Actin-1 selected as the housekeeping gene.Primer sequences are provided in the Appendix A.

Nuclear Localization of DAF-16
To determine DAF-16::GFP nuclear localization, synchronized L4 larvae of DAF-16::GFP-expressing TJ356 worms were randomly divided into control and ARE groups, which cultured on NGM plates containing 100 µg/mL ARE at 20 • C for 48 h.Then, the worms (n = 20) were transferred to a 2% agarose pad on a glass slide and anesthetized by 50 µg/mL levamisole.Fluorescence microscopy was used to capture and determine the location of DAF-16::GFP (cytoplasmic, intermediate between cytoplasm and nucleus, and nuclear).Representative images and number of DAF-16::GFP nuclear localization of C. elegans were obtained and counted.Statistical analysis was performed using the Student's t-test.The data are presented as mean ± SD.

Quantitation of GST-4::GFP and SOD-3::GFP Expression
The synchronized L4 stage of CF1553 and CL2166 worms were utilized to quantified the protein of SOD-3 and GST-4 expression, respectively.The CF1553 and CL2166 worms were incubated on NGM plates containing 100 µg/mL ARE at 20 • C for 2 days.Subsequently, the worms were anesthetized with 50 µg/mL levamisole hydrochloride and imaged using a fluorescence microscope (n = 20).

Screening and Identification of Active Substances from A. roxburghii
DPPH and ABTS assays were performed to evaluate the antioxidant capacity of A. roxburghii A, B, C, D, and ARE.The results demonstrated that 2 mg/mL ARE exhibited stronger scavenging abilities than 2 mg/mL A. roxburghii A, B, C, and D, reaching 83.55 ± 1.9% for DPPH and 64.11 ± 0.41% for ABTS (Figure 1a,b).The IC50 values of ARE for DPPH and ABTS were determined to be 0.244 mg/mL and 0.7681 mg/mL, respectively.The IC50 values for A. roxburghii A, B, C, and D can be found in Table A1.Therefore, ARE was selected and served as the primary active substance of A. roxburghii.Furthermore, UHPLC-MS/MS was employed to analyze the composition of ARE.The positive and negative total ion chromatograms of ARE are displayed in Figure 1c,d.After a detailed comparison of the mass-to-charge ratios (m/z) and secondary fragment information, a total of 65 compounds were identified in ARE (Table 1).tailed comparison of the mass-to-charge ratios (m/z) and secondary fragment information, a total of 65 compounds were identified in ARE (Table 1).

ARE Did Not Affect Food Selection, Proliferation, and Development of C. elegans
It was demonstrated that 100 µg/mL of ARE did not significantly affect OP50 proliferation (Figure 2a) and food choice of C. elegans (Figure 2b).Additionally, the average body length, egg laying, and movement were recorded to evaluate the impact of ARE on the growth and development of C. elegans.As shown in Figure 2c-e, the body length of C. elegans in the control, 10 of µg/mL ARE, and 100 µg/mL of ARE group was 1053.2 ± 85 µm, 1026.6 ± 52.7 µm, and 1041.3 ± 48.1 µm, respectively.The number of eggs laid by C. elegans in the control, 10 µg/mL of ARE, and 100 µg/mL of ARE group was 321.3 ± 56.1, 348.1 ± 28.6, and 311.7 ± 33.8, respectively.The average locomotion frequency of C. elegans was 12.6 ± 3.2 times, 12.6 ± 3.1 times, and 13.3 ± 4 times per 60 s in the control, 10 µg/mL of ARE, and 100 µg/mL of ARE group, respectively.These results reveal that ARE has no significant effect on the proliferation and development of C. elegans.

ARE Reduced the Accumulation of Lipofuscin and Prolonged the Life Span of C. elegans
As aging progresses, lipofuscin gradually accumulates in the intestines of C. elegans.As shown in Figure 3a,b, compared with the control group, the relative level of lipofuscin in C. elegans decreased to 66.6 ± 2.7% (p < 0.01) and 55.3 ± 2.8% (p < 0.01) after treatment with 10 µg/mL and 100 µg/mL ARE.The result of the survival experiment was displayed in Figure 3c.In the control group, the median lifespan of C. elegans was 17 days, with an average lifespan of 17.38 days and a maximum lifespan of 22 days.In the 10 µg/mL ARE group, the median lifespan was 19 days, with an average lifespan of 18.38 days and a maximum lifespan of 23 days, resulting in a 5.7% increase in average lifespan compared to the control group.In the 100 µg/mL ARE group, the median lifespan increased to 20 days, with an average lifespan of 20.21 days and a maximum lifespan extending to 27 days, representing a 16.3% increase in average lifespan compared to the control group.

ARE Reduced the Accumulation of Lipofuscin and Prolonged the Life Span of C. elegans
As aging progresses, lipofuscin gradually accumulates in the intestines of C. elegans.As shown in Figure 3a,b, compared with the control group, the relative level of lipofuscin in C. elegans decreased to 66.6 ± 2.7% (p < 0.01) and 55.3 ± 2.8% (p < 0.01) after treatment with 10 µg/mL and 100 µg/mL ARE.The result of the survival experiment was displayed in Figure 3c.In the control group, the median lifespan of C. elegans was 17 days, with an average lifespan of 17.38 days and a maximum lifespan of 22 days.In the 10 µg/mL ARE group, the median lifespan was 19 days, with an average lifespan of 18.38 days and a maximum lifespan of 23 days, resulting in a 5.7% increase in average lifespan compared to the control group.In the 100 µg/mL ARE group, the median lifespan increased to 20 days, with an average lifespan of 20.21 days and a maximum lifespan extending to 27 days, representing a 16.3% increase in average lifespan compared to the control group.ns-not significant.

ARE Reduced the Accumulation of Lipofuscin and Prolonged the Life Span of C. elegans
As aging progresses, lipofuscin gradually accumulates in the intestines of C. elegans.As shown in Figure 3a,b, compared with the control group, the relative level of lipofuscin in C. elegans decreased to 66.6 ± 2.7% (p < 0.01) and 55.3 ± 2.8% (p < 0.01) after treatment with 10 µg/mL and 100 µg/mL ARE.The result of the survival experiment was displayed in Figure 3c.In the control group, the median lifespan of C. elegans was 17 days, with an average lifespan of 17.38 days and a maximum lifespan of 22 days.In the 10 µg/mL ARE group, the median lifespan was 19 days, with an average lifespan of 18.38 days and a maximum lifespan of 23 days, resulting in a 5.7% increase in average lifespan compared to the control group.In the 100 µg/mL ARE group, the median lifespan increased to 20 days, with an average lifespan of 20.21 days and a maximum lifespan extending to 27 days, representing a 16.3% increase in average lifespan compared to the control group.

ARE Enhanced the Stress Resistance of C. elegans
After 0.1 J of UV irradiation, the average lifespan of C. elegans in the control group was 4.43 days, with a maximum survival time of 8 days.In the 10 µg/mL ARE group, the average lifespan was 4.51 days, with a maximum survival time of 8 days, showing no significant difference between the two groups.However, in the 100 µg/mL ARE group, the average lifespan increased to 5.08 days, representing a 14.7% increase (p < 0.01), with the maximum survival time extending to 9 days (Figure 3c).
At 37 • C, the average survival time of the control group was 7.82 h, with a maximum survival time of 15 h.In the 10 µg/mL ARE group, the average lifespan was 8.28 h, representing a 5.8% increase (p < 0.05), with a maximum survival time extending to 16 h.In the 100 µg/mL ARE group, the average lifespan was 9.04 h, representing a 15.6% increase (p < 0.01), with the maximum survival time extending to 17 h.These results indicate a significant improvement in the stress resistance of C. elegans treated with 100 µg/mL ARE (Figure 3d).

ARE Increased Antioxidant Enzyme Activity and Decreased ROS Accumulation in C. elegans
ROS accumulation in C. elegans was detected after treatment with ARE for 28 h.The results demonstrated that, compared to the control group, 10 µg/mL and 100 µg/mL of ARE significantly decreased the level of ROS in C. elegans (p < 0.01) (Figure 4a).elegans under 37 °C heat stress (n = 200).Statistical analysis was conducted using Student's t-test.Data from lifespan analysis were analyzed using Kaplan-Meier analysis and a log-rank test.Data are shown as mean ± SD. **** p < 0.0001, ns-not significant.

ARE Enhanced the Stress Resistance of C. elegans
After 0.1 J of UV irradiation, the average lifespan of C. elegans in the control group was 4.43 days, with a maximum survival time of 8 days.In the 10 µg/mL ARE group, the average lifespan was 4.51 days, with a maximum survival time of 8 days, showing no significant difference between the two groups.However, in the 100 µg/mL ARE group, the average lifespan increased to 5.08 days, representing a 14.7% increase (p < 0.01), with the maximum survival time extending to 9 days (Figure 3c).
At 37 °C, the average survival time of the control group was 7.82 h, with a maximum survival time of 15 h.In the 10 µg/mL ARE group, the average lifespan was 8.28 h, representing a 5.8% increase (p < 0.05), with a maximum survival time extending to 16 h.In the 100 µg/mL ARE group, the average lifespan was 9.04 h, representing a 15.6% increase (p < 0.01), with the maximum survival time extending to 17 h.These results indicate a significant improvement in the stress resistance of C. elegans treated with 100 µg/mL ARE (Figure 3d).

ARE Increased Antioxidant Enzyme Activity and Decreased ROS Accumulation in C. elegans
ROS accumulation in C. elegans was detected after treatment with ARE for 28 h.The results demonstrated that, compared to the control group, 10 µg/mL and 100 µg/mL of ARE significantly decreased the level of ROS in C. elegans (p < 0.01) (Figure 4a).The results of antioxidant enzyme activity showed that compared to the control group, 10 µg/mL ARE significantly increased SOD activity while having no significant effect on MDA content, CAT, and GSH-px activity (p > 0.05).Interestingly, 100 µg/mL ARE significantly decreased MDA and increased SOD, CAT, and GSH-px activity (p < 0.05).Specifically, as shown in Figure 4b-e, compared to the control group, 100 µg/mL ARE decreased MDA to 84.4 ± 6.4%, but increased SOD, CAT, and GSH-px activities to 242.7 ± 3.2%, 152.6 ± 15.7%, and 138.5 ± 2.7%, respectively (p < 0.01).

RNA-Seq and Enrichment Analysis
RNA-seq analysis was further performed to explore the potential differentially expressed genes (DEGs) and mechanisms of ARE in prolonging the lifespan of C. elegans.The OPLS-DA results showed that the control group and 100 µg/mL ARE group were well clustered (Figure 5a).Subsequently, compared to the control group, 581 differentially expressed genes (DEGs) were identified (p < 0.05, |log2FC| > 1), with 250 upregulated genes and 331 downregulated genes (Figure 5b,c).The results of GO enrichment analysis revealed that the biological process (BP) entries were predominantly linked to fatty acid beta-oxidation, xenobiotic metabolic processes, organic acid metabolic processes, and exogenous drug catabolic processes.Additionally, molecular function (MF) entries were identified and primarily associated with activities such as the structural composition of the cuticle, aromatase activity, heme binding, iron ion binding, UDP-glycosyltransferase activity, glucuronosyltransferase activity, and carboxylic ester hydrolase activity.The cellular components (CCs) entries were mainly linked to collagen trimers, organelle membranes, intracellular membrane-bounded organelles, peroxisomes, extracellular regions, and endoplasmic reticulum membranes.The BP, CC, and MF were displayed based on p value ranking (Figure 5d).Furthermore, KEGG pathway enrichment analysis further highlighted the alteration of 29 related signaling pathways, significantly enriching pathways involved in peroxisome, FoxO signaling pathway, TGF-beta signaling pathway, and Wnt signaling pathway.The top 20 signaling pathways are displayed in Figure 5e.These results indicate that the expression of antioxidant enzymes in the worms was activated, and ARE might Furthermore, KEGG pathway enrichment analysis further highlighted the alteration of 29 related signaling pathways, significantly enriching pathways involved in peroxisome, FoxO signaling pathway, TGF-beta signaling pathway, and Wnt signaling pathway.The top 20 signaling pathways are displayed in Figure 5e.These results indicate that the expression of antioxidant enzymes in the worms was activated, and ARE might extend the lifespan of C. elegans through the FoxO signaling pathway.

Metabolomics Analysis
Metabolomics analysis was further performed to explore the differential metabolites and mechanism of ARE in prolonging the lifespan of C. elegans.The OPLS-DA results showed that the control group and 100 µg/mL ARE group were well clustered (Figure 6a).In this study, 384 differential metabolites (263 upregulated and 121 downregulated) were identified based on the criteria of p < 0.05 and VIP > 1.0 (Figure 6b,c).Subsequently, the results of KEGG pathway enrichment analysis showed that the top 20 pathways mainly involved ABC transporters, arginine biosynthesis, alanine, aspartate and glutamate metabolism, aminoacyl-tRNA biosynthesis, the mTOR signaling pathway, glyoxylate and dicarboxylate metabolism, and the FoxO signaling pathway (Figure 6d).Coincidentally, the FoxO signaling pathway has also been identified in metabolomics after treatment with ARE in C. elegans.

Integration of Transcriptomics and Metabolomics Networks in C. elegans
The random forest method was used to screen the important biomarkers.The top 30 biomarkers were displayed in Figure 7a.Furthermore, as shown in Figure 7b, the top 20 genes and the top 20 metabolites were obtained and used to build correlation heatmaps.The association network and the number of association nodes were constructed and counted by integrating the absolute value of the correlation coefficient into the top 100 genes and metabolites (Figure 7c,d).As depicted in Figure 7e, we further constructed a differential network by integrating significantly altered genes and metabolites, subsequently mapping these entities to their corresponding pathways.As a result, 19 commonly affected pathways were identified (Figure 7f), including the following: arginine biosynthesis, biosynthesis of unsaturated faRy acids, FoxO signaling pathway, and mTOR signaling pathway.

Integration of Transcriptomics and Metabolomics Networks in C. elegans
The random forest method was used to screen the important biomarkers.The top 30 biomarkers were displayed in Figure 7a.Furthermore, as shown in Figure 7b, the top 20 genes and the top 20 metabolites were obtained and used to build correlation heatmaps.The association network and the number of association nodes were constructed and counted by integrating the absolute value of the correlation coefficient into the top 100 genes and metabolites (Figure 7c,d).As depicted in Figure 7e, we further constructed a differential network by integrating significantly altered genes and metabolites, subsequently mapping these entities to their corresponding pathways.As a result, 19 commonly affected pathways were identified (Figure 7f), including the following: arginine biosynthesis, biosynthesis of unsaturated fatty acids, FoxO signaling pathway, and mTOR signaling pathway.3.9.ARE Upregulated the Expression Levels of the daf-16, sod-3, and gst-4 Real-time quantitative PCR was used to verify the expression of key genes in the FoxO signaling pathway, including akt-1, akt-2, daf-16, sod-3, and gst-4 genes, identified through multiomics analysis.As shown in Figure 8a, compared to the control group, ARE significantly upregulated the expression levels of daf-16, sod-3, and gst-4 genes in C. elegans, while it did not significantly alter the expression levels of akt-1 and akt-2 genes.Specifically, the expression levels of daf-16, sod-3, and gst-4 genes increased to 290.8 ± 13.6%, 216.2 ± 17.4%, and 139.4 ± 20.7%, respectively, after treatment with ARE compared to the control group.
Subsequently, nematode TJ356 was used to evaluate the effects of ARE on the subcellular localization of daf-16.As shown in Figure 8b,c, the number of nuclear-localized daf-16 significantly increased in worms after treatment with ARE.Additionally, transgenic nematodes CF1553 and CL2166 were used to evaluate the translation of gst-4 and sod-3 genes downstream of daf-16 in C. elegans, respectively.The results of the CL2166 and CF1553 transgenic worms showed that sod-3::GFP expression increased to 250.1 ± 50.9% (p < 0.01) and GST-4::GFP expression increased to 133 ± 19.7% (p < 0.01) (Figure 8d-g).Subsequently, nematode TJ356 was used to evaluate the effects of ARE on the subcellular localization of daf-16.As shown in Figure 8b,c, the number of nuclear-localized daf-16 significantly increased in worms after treatment with ARE.Additionally, transgenic nematodes CF1553 and CL2166 were used to evaluate the translation of gst-4 and sod-3 genes downstream of daf-16 in C. elegans, respectively.The results of the CL2166 and CF1553 transgenic worms showed that sod-3::GFP expression increased to 250.1 ± 50.9% (p < 0.01) and GST-4::GFP expression increased to 133 ± 19.7% (p < 0.01) (Figure 8d-g).

ARE Prolongs the Lifespan of C. elegans through the daf-16/FoxO Pathway
To further clarify the role of ARE in promoting the expression of downstream genes sod-3 and gst-4 by upregulating daf-16, we used daf-16-deficient nematodes (CF1038) to evaluate the effect of ARE on the lifespan of C. elegans.The results showed that the lifespan of daf-16-deficient nematodes treated with ARE was not significantly different from that of the control group, confirming that ARE could prolong the lifespan of nematodes by promoting the expression of daf-16 (Figure 8h).

Discussion
In this study, we focused on A. roxburghii, an Asian plant with traditional medicinal and dietary uses, to explore its anti-aging properties in C. elegans.This research is the first to demonstrate that A. roxburghii can extend the lifespan of C. elegans.Using mutant nematode strains, transcriptomic, metabolomic, and other methods, we found that the activation of DAF-16/FoxO transcriptional nuclear activity, mediated by A. roxburghii, is crucial for the observed lifespan extension in C. elegans.
Previous studies have indicated that A. roxburghii possesses strong in vitro antioxidant activity [18,21], and our research has confirmed this finding.To further investigate the potential in vivo antioxidant activity of A. roxburghii, we used C. elegans as a model organism.The results showed that A. roxburghii extended the lifespan of C. elegans by  To further clarify the role of ARE in promoting the expression of downstream genes sod-3 and gst-4 by upregulating daf-16, we used daf-16-deficient nematodes (CF1038) to evaluate the effect of ARE on the lifespan of C. elegans.The results showed that the lifespan of daf-16-deficient nematodes treated with ARE was not significantly different from that of the control group, confirming that ARE could prolong the lifespan of nematodes by promoting the expression of daf-16 (Figure 8h).

Discussion
In this study, we focused on A. roxburghii, an Asian plant with traditional medicinal and dietary uses, to explore its anti-aging properties in C. elegans.This research is the first to demonstrate that A. roxburghii can extend the lifespan of C. elegans.Using mutant nematode strains, transcriptomic, metabolomic, and other methods, we found that the activation of DAF-16/FoxO transcriptional nuclear activity, mediated by A. roxburghii, is crucial for the observed lifespan extension in C. elegans.
Previous studies have indicated that A. roxburghii possesses strong in vitro antioxidant activity [18,21], and our research has confirmed this finding.To further investigate the potential in vivo antioxidant activity of A. roxburghii, we used C. elegans as a model organism.The results showed that A. roxburghii extended the lifespan of C. elegans by 16.3%, reduced ROS and lipofuscin accumulation, and had no adverse effects on the growth, development, or reproduction of the nematodes.In summary, these results support the effectiveness of A. roxburghii as an in vivo antioxidant and demonstrate its bioavailability.These findings are consistent with several other studies that have clearly demonstrated the antioxidant properties of A. roxburghii [21,26].
To elucidate the potential mechanisms underlying the anti-aging effects on C. elegans, differential gene and metabolite analysis revealed significant enrichment of the FoxO signaling pathway.The FoxO signaling pathway plays a crucial role in regulating various cellular processes such as the cell cycle, proliferation, apoptosis, and antioxidant stress response [27,28].In C. elegans, this pathway includes daf-2/IGFR, age-1/IP3K, and daf-16/FoxO [29].Studies have shown that the increased nuclear translocation of daf-16 is one of the main mechanisms for extending the lifespan of C. elegans.The nuclear translocation of daf-16 stimulates the expression of downstream antioxidant enzymes, thereby reducing the accumulation of oxidative damage [30][31][32].Treatment with ARE significantly increased the nuclear translocation of daf-16 in C. elegans and upregulated the expression of downstream antioxidant enzymes SOD-3 and GST-4.When the daf-16 was knocked out, the lifespan extension effect of ARE was abolished.Additionally, we found that ARE had no effect on the reproduction of C. elegans, confirming our hypothesis that reproductive signaling is not involved in ARE-induced lifespan extension.Therefore, we propose that ARE activates the FoxO signaling pathway, promotes the nuclear translocation of daf-16/FoxO transcription factors, and subsequently stimulates and regulates the expression of these two downstream proteins, SOD-3 and GST-4.
However, this study has some limitations.Firstly, it only demonstrated that ARE can activate daf-16 to extend the lifespan of C. elegans and its primary and secondary metabolites have been identified.The specific relationship between these metabolites and daf-16 remains unclear, including which substances play a role in extending lifespan and how they influence daf-16 expression to exert their effects.Additionally, further research is needed to investigate the potential of ARE in improving age-related diseases.
In conclusion, ARE demonstrates anti-aging effects on C. elegans and holds potential for developing anti-aging products, providing significant benefits for middle-aged and elderly populations.
Residue 1 was dried and subjected to ultrasonic extraction using 100 mL of 60% ethanol at 60 • C and 300 W for 1 h.This process was repeated five times, with the filtrates combined and concentrated by rotary evaporation, yielding residue 2. The ethanol extract was divided into supernatant and precipitate fractions by adjusting the pH to 2.0 using dilute hydrochloric acid and allowing it to stand in the refrigerator for 12 h, followed by centrifugation.After concentrating the supernatant through rotary evaporation at 60 • C, it was dried to obtain Extract B, weighing 1.13 g with a yield of 22.6%.Similarly, the precipitate was dried at the same temperature to yield Extract C, weighing 0.504 g with a yield of 10.08%.Extracts B and C primarily contain highly polar compounds, such as certain organic acids, phenolic compounds, and water-soluble flavonoids.
Residue 2 was dried at a low temperature and then subjected to Soxhlet extraction using 100% ethanol at a solid-liquid ratio of 1:20 for 1 h.Subsequently, the filtered drug residue 3 was extracted with boiling water (1:5 w/v) for 1 h.It was then mixed with four volumes of anhydrous ethanol, left to precipitate at 4 • C for 12 h, and the resulting precipitate was treated with a chloroform-butanol solution in a 1:4 ratio (v/v) for three washes before drying.This product was labeled as Extract D, yielding 0.012 g with an extraction rate of 0.24%.Meanwhile, the supernatant was concentrated by rotary evaporation at 60 • C, resulting in Extract E with a mass of 0.072 g and an extraction rate of 1.44%.Extracts D and E primarily contain moderately polar compounds, such as polysaccharides, proteins, certain alkaloids, and high-molecular-weight flavonoids.
freeze-drying, the A. roxburghii was ground into a fine powder for later use.Five grams the dried powder were weighed and subjected to Soxhlet extraction with 100 mL of p troleum ether at a 1:20 solid-liquid ratio for 18 h, yielding residue 1 and a petroleu ether extract.The petroleum ether extract, designated as Extract A, was concentrat using rotary evaporation and dried at 60 °C.The resulting mass of Extract A after dryi was 0.058 g, indicating an extraction yield of approximately 1.16%.This extract p dominantly comprises non-polar or weakly polar compounds, including fats, wax specific steroids, and non-polar aliphatic compounds.
Residue 1 was dried and subjected to ultrasonic extraction using 100 mL of 60 ethanol at 60 °C and 300 W for 1 h.This process was repeated five times, with the filtra combined and concentrated by rotary evaporation, yielding residue 2. The ethanol extr was divided into supernatant and precipitate fractions by adjusting the pH to 2.0 usi dilute hydrochloric acid and allowing it to stand in the refrigerator for 12 h, followed centrifugation.After concentrating the supernatant through rotary evaporation at 60 ° it was dried to obtain Extract B, weighing 1.13 g with a yield of 22.6%.Similarly, the p cipitate was dried at the same temperature to yield Extract C, weighing 0.504 g with yield of 10.08%.Extracts B and C primarily contain highly polar compounds, such certain organic acids, phenolic compounds, and water-soluble flavonoids.
Residue 2 was dried at a low temperature and then subjected to Soxhlet extracti using 100% ethanol at a solid-liquid ratio of 1:20 for 1 h.Subsequently, the filtered dr residue 3 was extracted with boiling water (1:5 w/v) for 1 h.It was then mixed with fo volumes of anhydrous ethanol, left to precipitate at 4 °C for 12 h, and the resulting p cipitate was treated with a chloroform-butanol solution in a 1:4 ratio (v/v) for thr washes before drying.This product was labeled as Extract D, yielding 0.012 g with extraction rate of 0.24%.Meanwhile, the supernatant was concentrated by rotary evap ration at 60 °C, resulting in Extract E with a mass of 0.072 g and an extraction rate 1.44%.Extracts D and E primarily contain moderately polar compounds, such as po saccharides, proteins, certain alkaloids, and high-molecular-weight flavonoids.Next, prepare a 0.1 mM DPPH solution in anhydrous ethanol.Mix 3 mL of the DPPH solution with 1 mL of each sample solution, vortex thoroughly, and incubate the mixture at room temperature in the dark for 30 min to allow the free radicals in the liquid to be fully scavenged.Then, measure the absorbance of the samples at 517 nm, using a water and ethanol mixture as a blank.Use glutathione as the positive control group.The scavenging activity is calculated using the following formula: A1 is the absorbance of 3 mL sample + 1 mL DPPH.A2 is the absorbance of 3 mL sample solvent + 1 mL DPPH.A3 is the absorbance of 3 mL sample + 1 mL ethanol.

Appendix A.3. ABTS Radical Scavenging Assay
Prepare the ABTS stock solution by mixing equal volumes of 7 mmol/L ABTS aqueous solution and 4.9 mmol/L potassium persulfate solution.Store the mixture in the dark.Dilute the ABTS stock solution to create a series of gradient dilutions.In a microplate, add 180 µL of each diluted ABTS solution and 20 µL of deionized water.Incubate the mixture at 37 • C in the dark for 5 min, then measure the absorbance at 405 nm.Select the dilution with an absorbance value around 0.7 as the ABTS working solution for subsequent experiments (prepare fresh as needed).
Add 20 µL of different concentrations of the extract sample solution to a microplate well, followed by 180 µL of ABTS working solution.Gently vortex to mix and incubate at 37 • C in the dark for 5 min.Measure the absorbance at 405 nm (A1).Use the sample solvent instead of the extract solution for the blank control (A2), and use distilled water instead of the ABTS working solution for the zero-adjustment control (A3).Use reduced GSH at the same concentration as the positive control group.The scavenging activity is calculated using the following formula:

Figure 1 .
Figure 1.Screening and identification of active substances from A. roxburghii.(a) DPPH scavenging capacity of different 2 mg/mL extracts.(b) ABTS scavenging capacity of different 2 mg/mL extracts.(c) Positive ion chromatogram of ARE.(d) Negative ion chromatogram of ARE.

Figure 1 .
Figure 1.Screening and identification of active substances from A. roxburghii.(a) DPPH scavenging capacity of different 2 mg/mL extracts.(b) ABTS scavenging capacity of different 2 mg/mL extracts.(c) Positive ion chromatogram of ARE.(d) Negative ion chromatogram of ARE.

Figure 2 .
Figure 2. Effects of ARE on OP50 and C. elegans.(a) Effect of ARE on the proliferation of OP50.(b) Preference of C. elegans for food containing ARE (n = 3).(c) The effect of ARE on the body length of C. elegans.(d) The effects of ARE on C. elegans reproduction (e) The effects of ARE on C. elegans locomotion.Statistical analysis was conducted using Student's t-test.Data are shown as mean ± SD. ns-not significant.

Figure 2 .
Figure 2. Effects of ARE on OP50 and C. elegans.(a) Effect of ARE on the proliferation of OP50.(b) Preference of C. elegans for food containing ARE (n = 3).(c) The effect of ARE on the body length of C. elegans.(d) The effects of ARE on C. elegans reproduction (e) The effects of ARE on C. elegans locomotion.Statistical analysis was conducted using Student's t-test.Data are shown as mean ± SD. ns-not significant.

Antioxidants 2024 , 20 Figure 2 .
Figure 2. Effects of ARE on OP50 and C. elegans.(a) Effect of ARE on the proliferation of OP50.(b) Preference of C. elegans for food containing ARE (n = 3).(c) The effect of ARE on the body length of C. elegans.(d) The effects of ARE on C. elegans reproduction (e) The effects of ARE on C. elegans locomotion.Statistical analysis was conducted using Student's t-test.Data are shown as mean ± SD. ns-not significant.

Figure 3 .
Figure 3. Effects of ARE on C. elegans.(a) The effect of ARE on the accumulation of lipofuscin in C. elegans (n = 20).Scale bar = 50 µm.(b) Effect of ARE on the lifespan of C. elegans (n = 200).(c) Effect of ARE on the lifespan of C. elegans under UV stress (n = 200).(d) Effect of ARE on the lifespan of C. elegans under 37 • C heat stress (n = 200).Statistical analysis was conducted using Student's t-test.Data from lifespan analysis were analyzed using Kaplan-Meier analysis and a log-rank test.Data are shown as mean ± SD. **** p < 0.0001.

Figure 3 .
Figure 3. Effects of ARE on C. elegans.(a) The effect of ARE on the accumulation of lipofuscin in C. elegans (n = 20).Scale bar = 50 µm.(b) Effect of ARE on the lifespan of C. elegans (n = 200).(c) Effect of ARE on the lifespan of C. elegans under UV stress (n = 200).(d) Effect of ARE on the lifespan of C.elegans under 37 °C heat stress (n = 200).Statistical analysis was conducted using Student's t-test.Data from lifespan analysis were analyzed using Kaplan-Meier analysis and a log-rank test.Data are shown as mean ± SD. **** p < 0.0001, ns-not significant.

Figure 4 .
Figure 4. ROS content and antioxidant enzyme activity.(a) Effect of different concentrations of ARE on ROS accumulation in C. elegans.(b) Effect of different concentrations of ARE on SOD enzyme

Figure 4 .
Figure 4. ROS content and antioxidant enzyme activity.(a) Effect of different concentrations of ARE on ROS accumulation in C. elegans.(b) Effect of different concentrations of ARE on SOD enzyme activity in C. elegans.(c) Effect of different concentrations of ARE on MDA content in C. elegans.(d) Effect of different concentrations of ARE on CAT enzyme activity in C. elegans.(e) Effect of different concentrations of ARE on GSH-px enzyme activity in C. elegans.Statistical analysis was conducted using Student's t-test.Data are shown as mean ± SD. * p < 0.05, ** p < 0.01, **** p < 0.0001, ns-not significant.

Figure 5 .
Figure 5. RNA seq and enrichment analysis E-100 µg/mL ARE; C-Control.(a) Orthogonal partial least squares discriminant analysis (OPLS-DA).(b) Volcano plot of significant DEGs (FC > 1.5, p < 0.05).(c) DEGs orange represents upregulation, while blue represents downregulation (FC > 1.5, p < 0.05).(d) DEGs expression GO analysis.(e) Differential gene expression KEGG analysis.The size of the circles corresponds to the number of DEGs and are color-coded according to −log10 (p value).The x-axis shows the enrichment factor value.

Figure 5 .
Figure 5. RNA seq and enrichment analysis E-100 µg/mL ARE; C-Control.(a) Orthogonal partial least squares discriminant analysis (OPLS-DA).(b) Volcano plot of significant DEGs (FC > 1.5, p < 0.05).(c) DEGs orange represents upregulation, while blue represents downregulation (FC > 1.5, p < 0.05).(d) DEGs expression GO analysis.(e) Differential gene expression KEGG analysis.The size of the circles corresponds to the number of DEGs and are color-coded according to −log10 (p value).The x-axis shows the enrichment factor value.

Figure 6 .
Figure 6.Identification of differential metabolites and enrichment analysis of metabolic pathways.E -100 µg/mL ARE.Ccontrol.(a) Orthogonal partial least squares discriminant analysis (OPLS-DA).(b) Volcano plot of differential metabolites.(c) Heatmap of differential metabolites.(d) KEGG pathway analysis of differential metabolites.The size of the circles corresponds to the number of DEMs and are color-coded according to p value.

Figure 6 .
Figure 6.Identification of differential metabolites and enrichment analysis of metabolic pathways.E-100 µg/mL ARE.C-control.(a) Orthogonal partial least squares discriminant analysis (OPLS-DA).(b) Volcano plot of differential metabolites.(c) Heatmap of differential metabolites.(d) KEGG pathway analysis of differential metabolites.The size of the circles corresponds to the number of DEMs and are color-coded according to p value.

Figure A2 .
Figure A2.We found that the fluorescence of lipofuscin in C. elegans began to weaken when the concentration of ARE reached 10 µg/mL, and there was no significant change in the decrease in lipofuscin when the concentration reached more than 100 µg/mL compared with 150 µg/mL.Therefore, we chose the two concentrations of 10 µg/mL and 100 µg/mL.Scale bar = 50 µm.

Figure A2 .
Figure A2.We found that the fluorescence of lipofuscin in C. elegans began to weaken when the concentration of ARE reached 10 µg/mL, and there was no significant change in the decrease in lipofuscin when the concentration reached more than 100 µg/mL compared with 150 µg/mL.Therefore, we chose the two concentrations of 10 µg/mL and 100 µg/mL.Scale bar = 50 µm.